The prevention of heparanase expression in endothelial cells injured by high glucose

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Vascular complications, in microvessels resulting in nephropathy, retinopathy and neuropathy and in macrovessels resulting in atherosclerosis caused by hyperglycemia contribute greatly to the morbidity and mortality in diabetes mellitus. In the vasculature, the endothelial cells (ECs) are first to be damaged by hyperglycemia due to their unique location as the inner lining of all vessels. There are several mechanisms involved in endothelial injury or dysfunction, however, the degradation of heparan sulfate proteoglycan (HSPG) on the cell surface and in the extra cellular matrix (ECM) is considered to be of importance. Heparanase is believed to degrade heparan sulfate (HS). Our objectives were to determine if heparanase is responsible for endothelial injury and dysfunction in diabetes. To determine if hyperglycemia and heparanase cause endothelial injury, high concentrations of glucose (30mM), mimicking hyperglycemia and optimal doses of heparinase I were used to treat cultured porcine aortic endothelial cells (PAECs). Cell injury was measured by determining live cell number and lactate dehydrogenase (LDH) release. To determine if heparanase is expressed in high glucose treated PAECs, reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify heparanase mRNA. In addition, heparanase activity was measured by incubating cell lysates with 35S-labelled ECM from cultured bovine corneal ECs, where released radioactive HS was analyzed by Sepharose gel filtration followed by â-scintillation counting. To help understand the mechanism of high glucose injury, heparanase mRNA and activity were also measured in PAECs treated with H2O2 or mannitol to determine if free radical injury or osmolarity caused effects similar to high glucose treatment. As well, high glucose or heparinase I treated PAECs were also treated with heparin (0.5 ìg/ml) and/or insulin (1 U/ml) and/or basic fibroblast growth factor (bFGF, 1 ng/ml) to determine if these compounds protected ECs from injury or inhibited heparanase expression induced by high glucose. p* PAECs injured by high glucose or heparinase I (0.3 U/ml in serum free medium) showed a significantly decreased live cell number and increased LDH release compared to control cells. High glucose or heparinase I treated ECs showed an increase in live cell number and decrease in LDH release when treated with heparin and/or insulin and bFGF. Heparanase mRNA and activity was expressed in PAECs treated with high glucose or H2O2. Heparin and/or insulin, but not bFGF prevented heparanase mRNA expression and activity in high glucose treated PAECs. Mannitol did not induce the upregulation of heparanase mRNA and activity. bFGF showed variable protection in cells treated with high glucose or heparinase I when combined with insulin or heparin. From these results we conclude that hyperglycemia is a main cause of endothelial injury. Heparanase production induced by hyperglycemia is responsible for EC injury and vascular dysfunction likely through the degradation of HS, resulting in increased vascular permeability and detachment of cells from the basement membrane. The mechanism of heparanase upregulation may be related to the formation of reactive oxygen species, but not due to changes in osmolarity. Heparin and/or insulin and bFGF protect cells from injury caused by high glucose or heparinase I. Heparin and/or insulin but not bFGF inhibit heparanase mRNA upregulation induced by high glucose. This study provides new insight into the causes of vascular injury associated with diabetes and suggests possible treatments to reduce endothelial injury.